U.S. patent number 5,120,740 [Application Number 07/542,664] was granted by the patent office on 1992-06-09 for prodrugs of 6-mercaptopurine and 6-thioguanine.
This patent grant is currently assigned to Wisconsin Alumni Research Foundation. Invention is credited to Adnan A. Elfarra.
United States Patent |
5,120,740 |
Elfarra |
June 9, 1992 |
**Please see images for:
( Certificate of Correction ) ** |
Prodrugs of 6-mercaptopurine and 6-thioguanine
Abstract
Prodrugs for the treatment of kidney tumors are disclosed. The
prodrugs are conjugates that can require the cooperation of
multiple enzymes in the kidney to release 6-mercaptopurine or
6-thioguanin, or can selectively release 6-thioguanine. Methods for
use of these prodrugs are also disclosed.
Inventors: |
Elfarra; Adnan A. (Madison,
WI) |
Assignee: |
Wisconsin Alumni Research
Foundation (Madison, WI)
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Family
ID: |
27029358 |
Appl.
No.: |
07/542,664 |
Filed: |
June 25, 1990 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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432098 |
Nov 3, 1989 |
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Current U.S.
Class: |
514/263.3;
544/276; 514/263.37; 544/265 |
Current CPC
Class: |
A61P
13/02 (20180101); A61P 15/00 (20180101); A61P
43/00 (20180101); C07D 473/38 (20130101); A61P
35/00 (20180101) |
Current International
Class: |
C07D
473/38 (20060101); C07D 473/00 (20060101); A61K
031/495 (); C07D 473/24 (); C07D 473/38 () |
Field of
Search: |
;514/262
;544/276,265 |
References Cited
[Referenced By]
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February 1959 |
Shive et al. |
2952539 |
September 1960 |
Dersch et al. |
3149111 |
September 1964 |
Hitchings et al. |
3232937 |
February 1966 |
Hitchings et al. |
3232938 |
February 1966 |
Hitchings et al. |
3238207 |
March 1966 |
Hitchings et al. |
3567705 |
March 1971 |
Cerny et al. |
4139705 |
February 1979 |
Dunbar et al. |
4169948 |
October 1979 |
Dunbar et al. |
4443435 |
April 1984 |
Bodor et al. |
4714701 |
December 1987 |
Beauchamp |
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Foreign Patent Documents
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350742 |
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Jan 1990 |
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EP |
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1695744 |
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Apr 1972 |
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DE |
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3730542 |
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Apr 1989 |
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DE |
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240663 |
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Oct 1987 |
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JP |
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Other References
Y Hwang et al., 251, J. Pharm. Exp., 448-454 (1989). .
L. Lash et al., 276, Arch. Bio. Biophys., 322-330 (1990). .
A. Garcia-Raso et al., 51, J. Org. Chem., 4285-4287 (1986). .
M. Winitz et al., 78, J. Amer. Chem. Soc., 2423-2430 (1956). .
FASEB Journal, Mar. 20, 1988, Abst. 4922, p. A1143, A. Elfarra et
al. .
FASEB Journal, Mar. 19, 1989, Abst. 1165, p. A427, A. Elfarra et
al. .
Vol. 30, Proc. A.A.C.R., Mar. 1989, p. 590, Abstract 2347, R.
Robins et al. .
Vol. 30, Proc. A.A.C.R., Mar. 1989, p. 596, Abstract 2374, J.
Fujitaki et al. .
Vol. 30, Proc. A.A.C.R., Mar. 1989, p. 597, R. Finch et al.
(Abstract 2375, 2377), R. Willis et al. (Abstract 2376), and G.
Crabtree et al. (Abstract 2378). .
Vol. 30, Proc. A.A.C.R., Mar. 1989, p. 599, Abstract 2384, T. Riley
et al. .
K. Van Scoik et al., 16, Drug Metab. Rev., 157-174 (1985). .
J. Nelson et al., 46, J. Can. Res., 137-140 (1986). .
Raz et al., Pharmacokinetics of Sulfur-35-Labeled
N-[8-(6-purinylthio)valeryl]glycine Ethyl Ester, Advan. Antimicrob.
Antineopl. Chemother. Proc. Int. Congress Chemother., 7th, 1971, 2,
59-61. .
Hwang, I. Y.; Elfarra, A. A., Cysteine S-Conjugates . . . , J.
Pharmacol. Exp. Ther., 1989, vol. 251(2)..
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Primary Examiner: Lee; Mary C.
Assistant Examiner: Gabilan; M. S. H.
Attorney, Agent or Firm: Quarles & Brady
Government Interests
This invention was made with U.S. government support awarded by
NIH:BRSG. The U.S. government has certain rights in this invention.
Parent Case Text
This application is a continuation in part of application Ser. No.
07/432,098 filed Nov. 3, 1989, now abandoned.
Claims
I claim:
1. A compound having the formula: ##STR8## where R.sub.1 is
selected from the group consisting of H and NH.sub.2 ; and
where R.sub.2 is selected from the group consisting of:
##STR9##
2. The compound of claim 1, wherein R.sub.1 is H and R.sub.2 is
##STR10##
3. The compound of claim 1, wherein R.sub.1 is H and R.sub.2 is
##STR11##
4. The compound of claim 1, wherein R.sub.1 is H and R.sub.2 is
##STR12##
5. The compound of claim 1, wherein R.sub.1 is NH.sub.2 and R.sub.2
is ##STR13##
6. The compound of claim 1, wherein R.sub.1 is NH.sub.2 and R.sub.2
is ##STR14##
7. The compound of claim 1, wherein R.sub.1 is NH.sub.2 and R.sub.2
is ##STR15##
8. A method of increasing the level of a compound selected from the
group of 6-mercaptopurine and 6-thioguanine in a mammalian kidney,
comprising the step of exposing the kidney to a compound having the
formula: ##STR16## where R.sub.1 is selected from the group
consisting of H and NH.sub.2 ; and
where R.sub.2 is selected from the group consisting of: ##STR17##
where R.sub.3 is alkyl, whereby said level is increased.
9. The method of claim 8 wherein R.sub.1 is H and R.sub.2 is
##STR18## and the level of 6-mercaptopurine is increased.
10. The method of claim 8 wherein R.sub.1 is H and R.sub.2 is
##STR19## and the level of 6-mercaptopurine is increased.
11. The method of claim 8 where R.sub.1 is H and R.sub.2 is
##STR20## and the level of 6-mercaptopurine is increased.
12. The method of claim 8 where R.sub.1 is H and R.sub.2 is
##STR21## and the level of 6-mercaptopurine is increased.
13. The method of claim 8 where R.sub.1 is H and R.sub.2 is
##STR22## and the level of 6-mercaptopurine is increased.
14. The method of claim 8 where R.sub.1 is NH.sub.2 and R.sub.2 is
##STR23## and the level of 6-thioquanine is increased.
15. The method of claim 8 where R.sub.1 is NH.sub.2 and R.sub.2 is
##STR24## and the level of 6-thioquanine is increased.
16. The method of claim 8 where R.sub.1 is NH.sub.2 and R.sub.2 is
##STR25## and the level of 6-thioquanine is increased.
17. The method of claim 8 where R.sub.1 is NH.sub.2 and R.sub.2 is
##STR26## and the level of 6-thioquanine is increased.
18. The method of claim 8 where R.sub.1 is NH.sub.2 and R.sub.2 is
##STR27## and the level of 6-thioquanine is increased.
19. A method of increasing the level of 6-thioguanine in a
mammalian kidney, comprising the step of exposing the kidney to a
compound, having the formula: ##STR28## where R.sub.1 is NH.sub.2 ;
and where R.sub.2 is ##STR29## whereby said level is increased.
20. A compound having the formula: ##STR30## where R.sub.1 is
selected from the group consisting of H and NH.sub.2 ; and
where R.sub.2 is selected from the group consisting of: ##STR31##
and wherein if R.sub.1 is H, the R.sub.2 is ##STR32##
21. The compound of claim 20, wherein R.sub.1 is H and R.sub.2 is
##STR33##
22. The compound of claim 20, wherein R.sub.1 is NH.sub.2 and
R.sub.2 is ##STR34##
23. The compound of claim 20, wherein R.sub.1 is NH.sub.2 and
R.sub.2 is ##STR35##
24. A method of increasing the level of 6-mercaptopurine in a
mammalian kidney, comprising the step of exposing the kidney to the
compound having the formula ##STR36## where R.sub.1 is H and
R.sub.2 is ##STR37## whereby said level is increased.
25. A method of increasing the level of 6-mercaptopurine in a
mammalian kidney, comprising the step of exposing the kidney to the
compound of claim 21, whereby said level is increased.
26. A method of increasing the level of 6-mercaptopurine in a
mammalian kidney, comprising the step of exposing the kidney to the
compound having the formula ##STR38## where R1 is H and R2 is
##STR39## whereby said level is increased.
27. A method of increasing the level of 6-thioguanine in a
mammalian kidney, comprising the step of exposing the kidney to the
compound having the formula ##STR40## where R.sub.1 is NH.sub.2 and
R.sub.2 is ##STR41## whereby said level is increased.
28. A method of increasing the level of 6-thioguanine in a
mammalian kidney, comprising the step of exposing the kidney to the
compound of claim 22, whereby said level is increased.
29. A method of increasing the level of 6-thioguanine in a
mammalian kidney, comprising the step of exposing the kidney to the
compound of claim 23, whereby said level is increased.
Description
This invention relates to compounds capable of targeting anti-tumor
drugs to the kidney. More specifically, it relates to prodrug
conjugates of 6-mercaptopurine and 6-thioguanine that are designed
to require exposure to multiple enzymes present in the kidney prior
to releasing an anti-tumor agent.
BACKGROUND OF THE INVENTION
The chemical modification of a biologically active drug to give a
new chemical from which the active drug can be generated by
enzymatic action is an important strategy to target drug action to
specific cells and tissues and thereby decrease toxicity or side
effects on non-target cells. N. Bodor et al., 22 Ann. Rep. Med.
Chem. 303-313 (1987); T. Krenitsky et al., 81 P.N.A.S. USA
3209-3213 (1984); J. Hjelle et al., 229 J. Pharmacol. Exp. Ther.
372-380 (1984); S. Magnan et al., 25 J. Med. Chem. 1018-1021
(1982); M. Orlowski et al., 212 J. Pharmacol. Exp. Ther. 167-172
(1980). The disclosure of these articles and of all other articles
listed herein are incorporated by reference as if fully set forth
herein. Such compounds are known as "prodrugs".
In A. Elfarra et al. FASEB Journal, Abstract 4922 (Mar. 20, 1988)
our laboratory reported that S-(6-purinyl)-L-cysteine, the cysteine
derivative of the anti-tumor and immunosuppressant drug
6-mercaptopurine, could be a potential prodrug. In this regard,
6-mercaptopurine is known to be effective in the treatment of
various types of tumors. However, it is not actively transported
from the G.I. tract, and is readily metabolized by xanthine oxidase
to generate biologically inactive metabolites. Thus, it must be
given in large doses for a long time to be effective. The concept
is that the .beta.-lyase needed to release the 6-mercaptopurine is
predominantly found in the kidney. Further, the kidney transport
system tends to concentrate amino acid derivatives. This
development is of great importance since currently there is no
satisfactory chemotherapy technique for treating kidney tumors.
While this S-(6-purinyl)-L-cysteine approach had some success, even
greater specificity is desired. Further, 6-mercaptopurine may have
certain disadvantages for some patients which renders use of
6-thioguanine alternatives desirable.
In separate work, our laboratory has reported that gamma-glutamyl
transpeptidase, cysteinyl glycine dipeptidase, and cysteine
conjugate .beta.-lyase are all present in the kidney and may
cooperatively act to release an S-(1,2-dichlorovinyl) thio moiety
from S-(1,2-dichlorovinyl glutathione). A. Elfarra et al., 35
Biochem. Pharm. 283-288 (1986). Also, it has been reported that the
renal acylase and the renal esterase are prevalent in the
kidney.
It has also been reported in A. Elfarra et al., 83 P.N.A.S. U.S.A.
2667, 2670 (1986) that a homocysteine S-conjugate of dichlorovinyl
could undergo transamination in the kidney to yield the
corresponding 2-keto acid, and that non-enzymatic elimination under
the conditions present in the kidney would then release the
dichlorovinyl group. The S-conjugate of the hydroxy analogue is
also toxic as it is metabolized to the keto-acid in the kidney by
amino acid oxidases. See L. Lash et al. 276 Arch. Biochem. Biophys.
322 (1990).
Thus, it can be seen that a need exists for the development of more
specific kidney prodrugs.
SUMMARY OF THE INVENTION
One embodiment of the present invention provides a compound having
the formula: ##STR1##
where R.sub.1 is selected from the group consisting of H and
NH.sub.2 ; and
where R.sub.2 is selected from the group consisting of:
##STR2##
In another aspect, the invention provides a compound having the
formula: ##STR3##
where R.sub.1 is selected from the group consisting of H and
NH.sub.2 ; and
where R.sub.2 is selected from the group consisting of:
##STR4##
In another aspect, the invention provides methods of inhibiting
tumor development in a mammalian kidney involving exposing the
kidney to one or more of these compounds.
Most of the above prodrugs require multiple enzyme activity prior
to releasing 6-mercaptopurine or thioguanine. In this regard, the
glutathione variants require action by gamma-glutamyl
transpeptidase (which releases a glutamate moiety), cysteinyl
qlycine dipeptidase (which releases a glycine residue), and
cysteine conjugate .beta.-lyase. The cysteinyl glycine variants
require use of the latter two enzymes to release the anti-tumor
agent. The acetyl variants require use of renal acylase and then
.beta.-lyase. The ester variants require use of renal esterase and
renal .beta.-lyase.
Because these five enzymes are predominantly found in the kidney,
because of the kidney's ability to concentrate amino acid
derivatives, and because significant concentrations of the
combination of these five enzymes are not found in many other parts
of the body, specificity can be achieved through use of these
compounds. Similarly, S-conjugates of homocysteine (and of hydroxy
analogues thereof) require enzymatic modification to the keto-acid
form, followed by further action in the kidney to release the
drug.
The objects of the invention therefore include:
(a) providing compounds of the above kind;
(b) providing methods of the above kind for using such compounds to
treat mammalian kidney tumors; and
(c) providing methods for synthesizing such compounds.
These and still other objects and advantages of the present
invention will be apparent from the description which follows.
These embodiments do not represent the full scope of the invention.
Rather, the invention may be employed in other embodiments.
Reference is therefore made to the claims herein for interpreting
the scope of the present invention.
BEST MODES FOR CARRYING OUT THE INVENTION
A detailed synthesis for S-(6-purinyl)-L-cysteine is described
below (where R.sub.1 =H and ##STR5## Synthesis of the claimed
compounds will then be described with reference to this synthesis.
Basically, 6-choroguanine or 6-chloropurine are reacted with the
amino acid precursor (glutathione, cysteinyl glycine,
N-acetyl-L-cysteine or cysteine alkyl ester, e.g. cysteine methyl
ester or cysteine ethyl ester) under the specified conditions to
yield the compound of interest. The compounds are then be tested on
rat kidney.
Materials
6-Chloropurine, 6-chloroguanine, glutathione, cysteinylglycine,
N-acetyl-L-cysteine, 6-mercaptopurine, and 2-chlorobenzothiazole
can be obtained from Aldrich Chemical Co. (Milwaukee, WI). Sephadex
LH-20 was supplied by Pharmacia Inc. (Piscataway, NJ). L-cysteine,
aminooxyacetic acid, cysteine methyl ester, cysteine ethyl ester,
and bovine serum albumin can be purchased from Sigma Chemical Co.
(St. Louis, MO). As standard for enzyme activity,
S-[2-benzylthiazolyl]-L-cysteine ("BTC") was prepared as previously
described D. Dohn et al., 120 Anal. Biochem. 379-386 (1982).
Supplies for proton NMR were obtained from Wilmad Glass Co. (Buena,
NJ). Other cysteinyl alkyl ester starting compounds can be prepared
using standard ester formulation reactions. All other reagents were
of the highest grade commercially available.
Synthesis Of S-6-Purinyl-L-Cysteine
6-Chloropurine (152 mg; 1.0 mmol) and L-cysteine (182 mg; 1.5 mmol)
were dissolved in a mixture of 2.5 mL of 1 N NaOH solution and 2.5
mL of methanol. This lead to the displacement of the chlorine by
the disodium salt of cysteine. The reaction mixture was kept under
nitrogen and heated in a water bath for 45 minutes at 50.degree. C.
with continuous stirring. The reaction mixture was kept in a ice
bath until S-6-purinyl-L-cysteine ("PC") was isolated by suing
either semi-preparative reverse phase HPLC or by low pressure
Sephadex LH-20 chromatography.
For the Sephadex LH-20 chromatography, the reaction mixture (pH
10.2-10.5) was applied to a 2.2.times.76 cm Sephadex LH-20 column
which was kept at 4.degree. C. because of the apparent instability
of PC when the Sephadex LH-20 chromatography was carried out at
room temperature. Product was separated from the reaction mixture
using water, adjusted to pH 10.5 with 1 N NaOH, as eluent. The flow
rate was 0.68 mL/minute and 5 minute fractions were collected using
a fraction collector. Fractions 55-62 which contained the product
were pooled, lyophilized and stored in a desiccator at -20.degree.
C. The yield of the compound was approximately 15%.
For the isolation of the compound by semi-preparative HPLC, samples
of the reaction mixture (adjusted to pH 5-6 with acetic acid) were
applied into a Whatman Partisil 10 ODS 3 magnum-9 HPLC column (9.4
mm.times.250 mm) attached to a Beckman 114M HPLC pump, a Rheodyne
injection valve, a 3 cm long C-I8 guard column, a Spectroflow 757
variable wavelength detector (Kratos Analytical Inc., NJ) and a
4270 SP Integrator (Spectra Physics Inc., NJ). The eluent was
acetonitrile:water (10:90 % v/v); flow rate was 2.0 mL/minute;
detection wavelength was 266 nm. The fractions that contained the
product (retention time 11-12 minutes) were collected and kept on
ice until it was lyophilized.
Analytical HPLC analyses were done as described for the
semi-preparative HPLC except that a 4.6.times.250 mm Whatman ODS 10
um column was used, and the flow rate was 1 mL/minute.
TLC were developed using 250 um Silicagel GF plates (Analtech Inc.,
DE) with isopropanol:water:ammonium hydroxide (9:2:1 5 v/v/v).
Product was visualized with a UV light (PC had a maximal UV
absorption at 284 nm; FIG. 3) or a ninhydrin spray. Rf values of
the product, 6-chloropurine, L-cysteine and L-cystine were 0.42,
0.67, 0.05 and 0.2 respectively with the indicated solvent
system.
Tests On Kidneys
Male Sprague-Dawley rats (250-350 g) (Charles River Laboratories,
Wilmington, MA), were killed by decapitation, and the kidneys were
removed. Subcellular fractionation by centrifugation was performed
as described by D. Dohn et al., 120 Anal. Biochem. 379-386 (1982).
These subcellular fractions were used as the enzyme source without
further purification. The protein concentrations of the different
fractions were determined according to the procedure of O. Lowry et
al., 193 J. Biol. Chem. 265-275 (1951) with bovine serum albumin as
the standard.
In a final volume 1.2 mL, each incubation tube contained sodium
borate buffer (pH 8.6, 0.1 M); the prodrug (3 mM); and the kidney
homogenate (4 mg/mL). Some tubes contained 1 mM aminooxyacetic
acid, an inhibitor of .beta.-lyase. Control incubations were
carried without the addition of the enzyme. Enzymatic reactions
were conducted for 20 minutes at 37.degree. C. in a Dubnoff
metabolic incubator with continuous shaking. Reactions were
terminated by placing the tubes on ice followed by filteration with
0.2 um Acro LC 13 membrane filters (VWR Scientific Inc., Chicago).
HPLC analyses were carried as described earlier with the additional
use of an authentic sample of 6-mercaptopurine as a reference.
Renal .beta.-lyase activity was determined with the prodrug as the
substrate and the amount of 6-mercaptopurine formed was determined
by HPLC. The assay mixture sodium borate buffer (pH 8.6; 0.1 M),
and enzyme solution (0.6-1.2 mg/mL). The reaction mixture was
incubated at 37.degree. C. for 20 minutes and the reaction was
terminated as described above. A portion (20 uL) of the filtrate
was injected into the HPLC system. The standard curve obtained by
plotting the peak area of the 6-mercaptopurine peak was linear over
the range of 1 to 350 uM of 6-mercaptopurine in the incubation
mixture.
To determine the effectiveness of the prodrug as a substrate for
renal .beta.-lyase, the rates of 6-mercaptopurine production were
compared with the rates of metabolism of the prototype .beta.-lyase
substrate, BTC (0.5-6 mM in 0.1 M sodium borate buffer, pH 8.6) to
form 2-mercaptobenzothiazole (D. Dohn et al., 120 Anal. Biochem.
379-386 (1982)).
My research showed that 6-mercaptopurine is generated by the
.beta.-lyase-dependent metabolism of the prodrug. Additional
studies showed that 6-mercaptopurine formation was dependent upon
incubation time, and substrate- and protein concentrations. The
results of the fractionation of various kidneys showed .beta.-lyase
activity in different subcellular fractions with the prodrug as the
substrate, and show that most of the prodrug dependent .beta.-lyase
activity was present in the cytosolic and mitochondrial fractions.
These results are in agreement with the subcellular localization of
renal .beta.-lyase.
The ability of the kidneys to concentrate amino acid derivatives
will result in the accumulation of prodrugs in renal proximal
tubular cells where it would be metabolized by .beta.-lyase (and
the other required enzymes) to form 6-mercaptopurine or
6-thioguanine. Thus, the kidneys will be exposed to high
concentrations of 6-mercaptopurine without exposing other portions
of the body to adverse concentrations of such compounds. This has
been verified by experiments in vivo on rats.
To synthesize the claimed compounds, one reacts 6-chloropurine or
6-chloroguanine with the precursor thio compound (L-cysteine
glutathione, cysteinyl-glycine, cysteine methyl ester, cysteine
ethyl ester, other cysteine alkyl ester, or N-acetyl-L-cysteine)
under similar conditions to yield
S-(6-purinyl)-N-acetyl-L-cysteine; S-(6-purinyl)-cysteinyl-glycine;
S-(6-purinyl)-glutathione; S-(6-purinyl)-cysteine methyl ester,
S-(6-purinyl)-cysteine ethyl ester, other S-(6-purinyl)-cysteine
alkyl esters; S-(6-guanyl)-N-acetyl-L-cysteine;
S-(6-guanyl)-cysteinyl-glycine; S-(6-guanyl)-cysteine methyl ester,
S-(6-guanyl)-cysteine ethyl ester; other S-(6-guanyl)-cysteine
alkyl esters; and S-(6-guanyl)-glutathione. S-(6-guanyl)-L-cysteine
can also be formed.
To create the homocysteine variants, one begins by making
S-(6-purinyl)-L-homocysteine (Compound Hl). To do this, sodium
metal is gradually added to a stirred solution of L-homocysteine
(Sigma Chemical Co.; 2 mmol) in liquid ammonia (150 ml) until a
blue color persisted for ten minutes. The white solid obtained
after the evaporation of the ammonia was redissolved in 15 ml water
containing EDTA (ethylene diamine tetraacetic acid; Aldrich
Chemical co.; 5 mg). 6-Chloropurine (2 mmol; Aldrich Chemical Co.)
was added and the pH of the solution was adjusted to 10.8 using a
10% solution of sodium hydroxide (Aldrich Chemical Co.). After
refluxing for 2.7 hours, the reaction mixture was cooled to room
temperature and the pH was adjusted to 2.7 using a 20% solution of
trifluoroacetic acid (Aldrich Chemical Co.). Portions of the
reaction mixture (2 ml each) were applied to a Pharmacia KX26
column, and the product was separated using a water (pH 2.5) as
eluent. The flow rate was 2.5 ml/min; UV-detection was at 254 nm;
Ve was 458 ml. The collects were pooled, lyophilized, and then
stored in a desicator.
Synthesis of the thioguanine variant (Compound H2) can be achieved
using 6-chloroguanine (Aldrich Chemical Co.) instead of
6-chloropurine. Synthesis of the homocysteine compounds where
R.sub.2 is ##STR6## (Compounds H3 and H4 respectively) will be
achieved by a modification of the method of M. Winitz et al., 78 J.
Amer. Chem. Soc. 2423-2430 (1956). One reacts sodium nitrite
(Aldrich Chemical Co.) with Compounds H1 and H2, respectively. The
synthesis of related compounds where R.sub.2 is ##STR7## can be
achieved by the reaction of Fremy's salt with Compounds H3 and H4,
respectively. See generally A. Garcia-Rafo et al. 51 J. Org. Chem.
4285 (1986) (Fremy's salt).
Further Testing
At 30 minutes after treatments of rats (in vivo) with
S-purinyl-L-glutathione (0.8 mmol/kg, i.p.), 6-mercaptopurine, and
its further metabolites 6-thiouric acid and 6-methylmercaptopurine,
were detected in the kidneys at concentrations of nearly 20 nmol
metabolites/g wet tissue. These compounds were not detected at all
in plasma or liver in this experiment (alneit with some dosages,
kill times, and other prodrugs small amounts of 6-mercaptopurine
can be detected in the plasma or liver).
At 24 hours after treatments of rats with
S-(purinyl)-L-glutathione, S-(purinyl)-N-acetyl-L-cysteine, or
S-(purinyl)-L-cysteine ethyl ester (0.4 mmol/kg, i.p.),
6-mercaptopurine and its metabolites were present in urine at
levels corresponding to 4, 2, and 1% of the theoretically possible
administered dose, respectively.
When rats were given S-(6-purinyl)-homocysteine (0.4 mmol/kg), the
amounts of 6-mercaptopurine and the products of its further
metabolism which were detected in urine at 24 hour post treatment
were at levels corresponding to four times that amount found for
S-(6-purinyl)-L-cysteine.
When rats were given the homocysteine variant (0.8 mmol/kg, i.p.)
the concentration of metabolites (6-mercaptopurine,
6-methylmercaptopurine, and 6-thiouric acid) present in liver and
kidneys at 30 minute post treatment were nearly 10 and 90 nmol/g
wet tissue, respectively. No metabolites were detected in plasma.
These results show that this compound is a selective kidney
precursor of 6-mercaptopurine.
This concept of providing a multiplicity of enzymes to control
release 6-mercaptopurine or 6-thioguanine provides a highly
advantageous system, and yields prodrugs with improved specificity.
Also, thioguanine prodrugs are highly desirable.
It will be appreciated that the above describes only the preferred
embodiments of the invention. A number of other modifications and
changes are within the scope of the invention and are intended to
be included within the scope of the claims. For example, DL (as
distinguished from L stereochemistry is included). Also,
equivalents such as radio labelled variants are included. Thus, the
claims should be looked to in judging the scope of the
invention.
* * * * *